Process for preparing collagen beads

ABSTRACT

Collagen beads prepared by adding spherical droplets of an aqueous liquid of collagen to an aqueous solution of tannin. The collagen beads show a uniform grain size and have either a two-layered structure consisting of an aqueous liquid sustained inside a solidified outer surface or a completely solidified form.

TECHNICAL FIELD

This invention relates to collagen beads and a process for producing thesame.

BACKGROUND OF THE INVENTION

Bead-type shaped articles produced by using biopolymers (for example,proteins, polysaccharides and the like) have been utilized as bead foodsand as supports for immobilizing physiologically active substances.

As a bead-type food, for example, Japanese Patent Laid-Open No.79755/1976 proposes a process for producing a fish roe-like food whichcomprises gelatinizing cold-gelling and edible aqueous sol droplets (forexample, gelatin) by cooling, further coating the gelatinized particlesthus formed with a film-forming sol and contacting the same with agelling agent to thereby form a gelatinized film layer. Further,Japanese Patent Publication No. 11101/1970 proposes to providegelatinized alginate particles by treating an alginate with a divalentmetal ion which can be incorporated into the body of a living organism.

As a bead-type shaped article produced with the use of collagen,Japanese Patent Publication No. 1169/1989 describes a process forproducing collagen beads which are usable as a support for immobilizinga physiologically active substance. This process comprises formingcollagen spheres in an organic solvent which is hardly miscible orimmiscible with water and then hardening the spheres.

However there has been developed no bead-type shaped collagen articlewhich is applicable to foods.

In the conventional methods for producing collagen beads as describedabove, an aqueous liquid of a high concentration of a collagen materialis employed and a hardly miscible or immiscible organic solvent is usedduring the production process. In general, many hardly miscible orimmiscible organic solvents are harmful to human beings. Therefore, theproduction of beads with the use of such organic solvents suffers fromproblems of the contamination and remaining of the organic solvents inthe beads. In the conventional methods, furthermore, aldehydes and metalsalts such as chromium salts are used in order to harden or crosslinkcollagen. Therefore, the beads thus obtained are hardly applicable tofoods.

SUMMARY OF THE INVENTION

In the present invention, no organic solvent but an aqueous liquid of acollagen material of a low concentration is used and, therefore, thebeads are never contaminated with any organic solvent. Further, asubstance used in the present invention for hardening the beads thusformed has a high degree of safety when applied to foods. Furthermore,the present invention makes it possible to produce collagen beads in alarge amount industrially.

The present inventors have conducted extensive studies in order toprovide a highly safe solution wherein the employed solvent is otherthan a hardly miscible or immiscible organic solvent, and an aqueousliquid of a collagen material can be formulated into spherical beadssimply by dropping the aqueous collagen liquid into said solvent. As aresult, they have successfully found out that spherical collagen beadscan be formed by dropping an aqueous liquid of a collagen material intoan aqueous solution of tannin which is a natural organic compoundfrequently contained in, for example, foods and drugs. They have furtherfound out that when the collagen beads thus formed are maintained in theaqueous solution of tannin, tannin penetrates into the sphericalcollagen beads to thereby solidify the collagen beads.

On the basis of these findings, the present inventors have discussed theconcentration of the collagen material in the aqueous liquid of thecollagen material, the concentration of tannin in the aqueous tanninsolution and the pH value of the aqueous tannin solution, each requiredfor the formation of the collagen beads, thus completing the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Collagen is a protein hardly soluble in water which constitutes animalconnective tissues such as bone and skin, and is in the form of rodswith a helical structure consisting of three peptide chains (molecularweight: 300,000, length: 300 nm, diameter: 1.5 nm). Different fromgelatin obtained by denaturing collagen, collagen is characterized bynot setting into gel, i.e. gelling, even at a low temperature.

As the aqueous liquid of a collagen material to be used as the startingmaterial in the present invention, collagen which has been solubilizedby treating with an enzyme or an alkali (refer to Japanese PatentPublication No. 11037/1969 and No. 15033/1971) may be cited. To formcollagen beads, the concentration of the collagen material in theaqueous liquid of the collagen material ranges from about 0.1 to about2.0% by weight. The tannin to be used in the present invention is notparticularly restricted. Namely, it may be arbitrarily selected fromamong those satisfying the common definition of tannin, i.e., "awater-soluble, polyphenol compound originating from a plant and becomingturbid or giving a precipitate in a solution together with proteins oralkaloids".

The concentration of the aqueous tannin solution may be at such a levelas to maintain the aqueous liquid of the collagen material in the formof spheres therein. That is to say, it may range from about 0.1 to about10.0% by weight, preferably from about 0.5 to about 5% by weight.Further, the pH value of the aqueous tannin solution may fall within arange wherein tannin is not oxidized. It usually ranges from about pH 2to about pH 7, preferably from about pH 2 to about pH 4.

As described above, tannin would generally form a precipitate togetherwith a protein in a solution. However, the collagen material to be usedas the starting material in the present invention would not becometurbid within the concentration range of the collagen material asspecified above. When an aqueous solution of the collagen material wasdropped into an aqueous tannin solution, however, it forms a precipitatein the form as it is. When the concentration of the collagen material inthe aqueous liquid thereof is higher than the level as specified above,therefore, the aqueous liquid of the collagen material dropped into theaqueous tannin solution would solidify not in the form of spheres but inthe form of droplets as it is, because of the high viscosity of theaqueous liquid. When the concentration of the collagen material is lowerthan the level as specified above, on the other hand, the aqueous liquidof the collagen material dropped into the aqueous tannin solution woulddiffuse into small pieces, thus becoming turbid similar to otherproteins.

The particle size of the collagen beads can be arbitrarily determined byvarying the droplet size, the concentration of the collagen material inthe aqueous liquid thereof, the concentration of tannin in the aqueoussolution thereof (i.e., the immersion liquid), etc. For example, when anaqueous liquid of a collagen material at a concentration of 0.1 to 2.0%by weight is dropped into an aqueous tannin solution at a concentrationof 0.1 to 10.0% by weight through a nozzle of 0.5 mm in inner diameter,the particle size of the collagen beads thus formed can be varied withina range of from 2.0 to 4.0 mm by controlling the concentrations of thecollagen material and tannin. When the aqueous liquid of the collagenmaterial is dropped into the aqueous tannin solution through a nozzle of3 mm in inner diameter, the particle size can be similarly varied withina range of from 3.0 to 8.0 mm.

According to the present invention, droplets comprising the sphericalaqueous liquid of the collagen material can be formed by dropping theabove-mentioned aqueous liquid of the collagen material into the aqueoustannin solution. Then the aqueous tannin solution is allowed to standunder slowly stirring. Thus the tannin penetrates into the sphericalcollagen beads and solidifies collagen therein. Further, the surface andinternal structures of the beads can be changed by controlling thepenetration time. When the immersion time is from 30 seconds to 30minutes, collagen beads of a two-layered structure, wherein the externalsurface solidifies while the content remains in the form of the aqueousliquid differing in the degree of solidification, are usually obtained,though this phenomenon depends on the concentration of the collagenmaterial and the concentration of tannin in the immersion aqueoussolution. When the immersion is performed for 30 minutes or longer,collagen beads which have been uniformly and completely solidified canbe obtained. That is to say, the ratio of tannin to the collagenmaterial in the collagen beads according to the present invention can bearbitrarily varied by controlling the concentration of the aqueoustannin solution and the immersion time. When a double-walled coaxialnozzle is used and the above-mentioned aqueous liquid of the collagenmaterial is extruded into the outer part of the coaxial nozzle whilevarious aqueous liquids and oily substances are extruded into the innerpart thereof at the same time followed by dropping into the aqueoustannin solution, furthermore, collagen beads consisting of a solidifiedouter surface and a content comprising a liquid or a gel of thesubstances other than collagen enclosed therein can be prepared.

Further, substances other than collagen may be mixed with the aqueousliquid of the collagen material. In this case, collagen beads havingsaid substances enclosed therein can be obtained. For example, collagenbeads can be prepared by mixing 5 parts by weight of a collagen materialwith 1 part by weight of dextran (molecular weight distribution: about60,000 to about 90,000) and dropping the obtained mixture into a 10.0%by weight aqueous tannin solution. When these collagen beads are allowedto stand in a solution of pH 4.0 for 5 hours, the amount of the dextranreleased from the collagen beads is not more than 0.1%. However, thesubstances capable of being enclosed in these beads are restricted topolymers having a molecular weight of at least about 60,000. Preferableexamples thereof include water-soluble proteins and polysaccharides suchas dextran as described above, BSA, starches, pectin and carrageenan,though substances which are hardly soluble or insoluble maybe used solong as they can be fully diffused in the aqueous solution of collagenwithout undergoing coagulation or separation. In contrast, low molecularweight substances such as amino acids and mono-, di- oroligo-saccharides can freely permeate through collagen beads.Accordingly, collagen beads containing a low molecular weight substancecan be produced by immersing collagen beads in a solution of such asubstance and equilibrating the beads and the solution.

The present invention aims at providing collagen beads applicable tofoods and a process for producing the same. The collagen beads of thepresent invention, which are prepared substantially from collagen andtannin, can be suitably used in foods, different from the conventionalones.

Namely, these collagen beads are applicable to foods, for example, afish roe-like food consisting of an outer coating and an inner solutionenclosed therein and confectionery. In these cases, the beads maycontain substances other than collagen such as coloring matters andflavorings. Regarding coloration, in particular, collagen beads of agray or black color can be obtained by immersing in a solutioncontaining ferric ions, since the beads consist of collagen and tannin.These products thus obtained are usable as a caviar-like food.

In addition, the collagen beads of the present invention, which are freefrom heavy metals, are usable as a support on which physiologicallyactive substances such as enzymes are immobilized without beinginactivated.

To further illustrate the present invention in greater detail, thefollowing Examples will be given. However, it is to be understood thatthe present invention is not restricted thereto.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

An aqueous liquid of enzymatically solubilized collagen (pH 5.0,concentration: 1.0% by weight) was sufficiently degassed under reducedpressure. Then it was dropped into an aqueous solution of tannic acid(pH 5.0, concentration: 1.0% by weight) through a nozzle of 0.5 mm indiameter with the use of a peristaltic pump at a flow rate of 2.0ml/min. After the completion of the addition, the aqueous solution oftannic acid was allowed to stand under slow stirring for 2 hours and wasthen sufficiently washed with distilled water. Thus uniform collagenbeads of 2.8±0.1 mm in diameter were obtained.

EXAMPLE 2

An aqueous liquid of enzymatically solubilized collagen (pH 8.0,concentration: 0.54 by weight) was sufficiently degassed under reducedpressure. Then it was dropped into an aqueous solution of grape seedtannin (pH 3.0, concentration: 7.5% by weight) through a nozzle of 0.5mm in diameter with the use of a peristaltic pump at a flow rate of 2.0ml/min.

The grape seed tannin was isolated in the following manner. Namely, 100g of grape seeds were ground and sufficiently defatted with the use ofhexane. Next, polyphenol was extracted by using 100 ml of 50% ethanol.After concentrating the obtained extract under reduced pressure tothereby eliminate the ethanol, the residue was centrifuged and theprecipitate was removed. To the obtained supernatant, was added diethylether. After thoroughly stirring, the aqueous layer was collected andfreeze-dried. The powder thus obtained was used as the grape seedtannin.

After the completion of the addition, the aqueous solution of grape seedtannin was allowed to stand under slow stirring for 2 hours. Thencollagen beads were taken out from the aqueous solution of grape seedtannin and thoroughly washed with distilled water. Thus uniform collagenbeads of 2.4±0.1 mm in diameter were obtained.

EXAMPLE 3

An aqueous liquid of enzymatically solubilized collagen (pH 3.0,concentration: 1.0% by weight) was mixed with 1.0% by weightalkali-treated gelatin at a ratio of 1:1 at 30° C. and then defoamedunder reduced pressure. Then it was dropped into an aqueous solution oftannic acid (pH 4.0, concentration: 3.0% by weight) through a nozzle of0.7 mm in diameter with the use of a peristaltic pump. Within 1 minuteafter the completion of the addition, collagen beads were taken out fromthe aqueous solution of tannic acid and immersed in a 10% solution ofsodium chloride for 2 hours. Thus, a food having a particle size of5.2±0.1 mm was obtained that had a fish roe-like texture with aninternal sol phase.

EXAMPLE 4

An aqueous liquid of enzymatically solubilized collagen (pH 3.0,concentration: 0.5% by weight) was mixed with soybean oil at a ratio of3:1 and emulsified. The obtained emulsion was then dropped into anaqueous solution of tannic acid (pH 3.0, concentration: 5.0% by weight)through a nozzle of 0.5 mm in diameter with the use of a peristalticpump. Within 2 minutes after the completion of the addition, beads weretaken out from the aqueous solution of tannic acid, colored in gray byimmersing in a 0.5% solution of iron chloride for 30 seconds and thenimmersed in a 5.0% solution of sodium chloride for 2 hours. Thus acaviar-like food being similar to caviar in color tone, taste andtexture were obtained.

EXAMPLE 5

To 50 ml of an aqueous liquid of alkali-solubilized collagen (pH 3.0,concentration: 2.0% by weight), was added 5 mg (2,000 U) of invertase,i.e., a sugar hydrolase and dissolved therein by mixing. After defoamingby using a cold-centrifuge, the mixture was introduced into an injectiontube and dropped into an aqueous solution of tannic acid (pH 3.0,concentration: 2.0% by weight) through a syringe of 0.2 mm in innerdiameter. After the completion of the addition, the aqueous tanninsolution was allowed to stand at 4° C. under slow stirring for 4 hoursand was then thoroughly washed with an aqueous solution (pH 3.0). Thuscollagen beads (particle size: 2.5±0.1 mm) on which the invertase hadbeen immobilized were obtained. When the enzymatic activity of theinvertase immobilized on these collagen beads was determined with theuse of sucrose as the substrate in a solution of pH 4.0, it was foundthat the enzyme fully sustained its activity.

What is claimed is:
 1. A process for producing collagen beads whichcomprises adding spherical droplets of an aqueous liquid of a collagenmaterial, containing about 0.1 to about 2.0% by weight of collagen, toan aqueous tannin solution to thereby form spherical solidified collagenbeads.
 2. A process as claimed in claim 1 wherein said aqueous liquid ofcollagen material is solubilized collagen.
 3. A process as claimed inclaim 1 wherein the tannin concentration of said aqueous tannin solutionranges from about 0.1 to about 10% by weight and the pH value of saidsolution ranges from about 2 to about
 7. 4. A process according to claim3 wherein said tannin concentration ranges from about 0.5 to about 5% byweight, and said pH value ranges from about 2 to about
 4. 5. A processas claimed in claim 1, wherein the tannin concentration of said aqueoustannin solution ranges from about 0.1 to about 10% by weight and the pHvalue of said solution ranges from about 2 to about
 7. 6. A methodaccording to claim 1 comprising controlling the droplet size so as toform said spherical solidified beads having a particle size of from 2.0to 8.0 mm.
 7. A process according to claim 1 further comprising slowlystirring said aqueous tannin solution after addition of said sphericaldroplets.
 8. A process according to claim 1 comprising maintaining saiddroplets in said aqueous tannin solution for from 30 seconds to 30minutes to form said spherical solidified beads having a two-layeredstructure comprising a solid external surface and a liquid interior. 9.A process according to claim 1 comprising maintaining said sphericaldroplets of collagen in said aqueous tannin solution for at least 30minutes to form said spherical solidified beads in completely solidifiedform.
 10. A process according to claim 1 further comprising contactingsaid spherical solidified beads with a ferric ion solution, a sodiumchloride solution, or both a ferric ion solution and a sodium chloridesolution.
 11. A process according to claim 1 wherein said aqueous liquidof a collagen material containing from about 0.1 to about 2.0% by weightof collagen further comprises a polymer having a molecular weight of atleast about 60,000 and which is fully diffused in said aqueous liquid ofcollagen.
 12. A process according to claim 1 wherein said aqueous liquidof a collagen material containing from about 0.1 to about 2.0% by weightof collagen further comprises invertase.
 13. A process for producingcollagen beads, comprising:forming an aqueous liquid consistingessentially of a collagen material having a collagen concentration fromabout 0.1 to about 2.0% by weight; providing an aqueous tannin solution;adding said aqueous liquid consisting essentially of collagen materialdropwise to said aqueous tannin solution thereby providing droplets ofcollagen in said aqueous tannin solution; and maintaining said collagendroplets in said aqueous tannin solution for a time sufficient tosolidify at least an outer layer of said collagen droplets and thus formcollagen beads.